This invention relates to immunological methods and devices for detecting analytes in biological samples.
Numerous approaches have been developed for detection of a given analyte in a biological sample. Typical of these methods are the so called "lateral flow" and "flow-through" devices and methods. The flow-through device generally uses a porous material with a reagent-containing matrix layered thereon or incorporated therein. Test sample is applied to and flows through the porous material, and analyte in the sample reacts with the reagent(s) to produce a detectable signal on the porous material. These devices are generally encased in a plastic housing or casing with calibrations to aid in the detection of the particular analyte.
Lateral flow assays also utilize a porous membrane for performing analyte detection. Instead of drawing the sample through the membrane perpendicularly, the sample is permitted to flow laterally from an application zone to a reaction zone on the membrane surface. The capture reagent is present in the reaction zone, and the captured analyte can be detected by a variety of protocols, including direct visualization of visible moieties associated with the captured analyte.
One-step lateral flow assays permit a user to add a sample to a sample application region and obtain a positive or negative signal signaling the presence or absence of the test analyte in the sample.
One-step lateral flow devices contain a sample application region to which the sample is applied. The sample application region is in lateral flow contact with the porous carrier material of the analyte detection region. During lateral flow, the sample is brought into contact with a mobile indicator reagent in a discrete zone of the analyte detection region. The indicator reagent contains both a binding moiety which specifically binds to the target analyte and an indicator moiety, which is most often a chromophore label. Target analyte molecules moving in the lateral flow bind to the indicator reagent and are ultimately immobilized in the capture zone, usually by binding to a second reagent which binds specifically to the analyte or to the analyte-indicator reagent complex. The position of the immobilized indicator reagent gives rise to a positive signal. Additional signals may include a negative reaction indicator, a test complete indicator, and a positive control indicator.
One-step immunochromatographic devices containing the indicator reagent in a discrete zone of the lateral flow porous material, e.g., at a discrete site on the test strip, have been described.
For example, Deutsch et al. describe a quantitative chromatographic test strip device in U.S. Pat. Nos. 4,094,647, 4,235,601 and 4,361,537. The device comprises a strip of material capable of transporting a solution by capillary action, i.e., wicking. Different areas or zones in the strip contain the reagents needed to produce a detectable signal as the analyte is transported to or through such zones. A diffusible label which can bind to the test analyte may be located in a discrete region of the strip. The device is suited for both chemical assays and binding assays which are typified by the binding reaction between an antigen and its complementary antibody.
In addition, British Application No. 2,204,398 describes a lateral flow device wherein sample applied to the device picks up labeled reagent located at a discrete site on the porous carrier of the strip and permeates into a detection zone. The indicator labels include gold sols and colored particles.
Alternatively, devices containing the mobile indicator reagent in a separate porous material or pad have been disclosed.
For instance, European Publication No. 323,605 discloses an assay device using chromatographic material wherein the test sample can travel from one end to the other by capillary action. The chromatographic material contains an immobilized capture reagent capable of binding to the analyte. The application pad which receives the test sample also contains a diffusible indicator reagent capable of migrating from the application pad to the chromatographic material. The indicator reagent is capable of binding to the analyte. The binding of the indicator reagent-analyte complex results in a detectable signal at the capture situs.
PCT application No. WO 94/06013 also describes a lateral flow assay in which the indicator reagent has been placed in a separate indicator reagent region or pad (referred to as "the third liquid permeable material"). The sample is added to a separate sample application pad, passes through a second permeable material, and mobilizes the indicator reagent located in the third liquid permeable material. The sample then enters the wicking material containing the capture zone.
Patent application Wo 92/01226 describes a lateral flow device in which the labeled specific binding reagent is retained in the dry state either in a zone on the carrier material Ao in a separate porous body through which the sample passes en route to the porous carrier material of the test strip.
U.S. patent application Ser. No.08/444,238 and its corresponding PCT application 99/04748 also describe lateral flow assay devices in which the labeled reagent for the analyte is located in a discrete zone of the porous carrier material of the analyte detection region.
Other variations of test strip assays are disclosed in U.S. Pat. Nos. 4,298,688, 4,517,288 and 4,740,468, which describe sheet-like diagnostic devices comprising one or several strips, arranged behind one another, having zones situated one behind another. Each zone is readily accessible from above and below for the addition of reagents. Such devices can quantitatively determine the amount of an analyte.
Procedures using chromogenic and fluorescent dyes as labels in biological assay procedures are also known. Typical assay protocols call for direct or indirect binding of a dye label to an analyte or analyte analog in a biological sample, where the presence or absence of the dye at a particular stage of the assay can be determined visually and related to the amount of analyte initially present in the sample. A wide variety of specific assay protocols exist.
A number of those assays utilize naturally colored or dyed particles as a label, where the particles are bound to an antibody or other specific binding substance. Suggested particles include dyed latex beads, dye imbibed liposomes, erythrocytes, metal sols, and the like. The colored particle in such complexes can serve as a visible marker, where separation, capture, or aggregation of the particles is mediated through binding of the antibody or other specific binding substance. The amount of label thus segregated in a particular assay step is related to the amount of analyte initially present in the sample.
For example, U.S. Pat. No. 4,943,522 describes a solid phase lateral flow assay using erythrocytes as a label. U.S. Pat. No. 4,863,875 describes compositions comprising at least ten dye molecules or monomers covalently attached to an antibody through an isocyanate group on the dye. U.S. Pat. No. 4,703,017 describes a solid phase assay device which relies on specific binding of a ligand-label conjugate on a solid support, where the label is disclosed as a particle, such as a liposome, or polymer microcapsule. U.S. Pat. No. 4,608,246 describes assays for typing blood which employ erythrocytes as a labeling agent. U.S. Pat. No. 20 4,452,886 describes the covalent attachment of photon absorbing or emitting polymers to proteins, such as antibodies and antigens. U.S. Pat. No. 4,373,932 describes labeling of a ligand with an aqueous dispersion of a hydrophobic dye or pigment, or a polymer nuclei coated with such a dye or pigment. U.S. Pat. No. 4,313,734 describes methods of detecting sample analytes by the determination of the metallic label content in the sample. U.S. Pat. No. 4,169,138 describes immunoassays which employ visible particles including undyed microorganisms, bound to polymers which may be of microbial origin.
Other lateral flow protocols include U.S. Pat. No. 4,943,522 directed to a lateral flow device which relies on a nonbibulous support to conduct liquids from one portion of the device to another. PCT Publication WO 92/12428, which is related to the above patent, represents an improvemetnt on that method and device wherein nonbibulous lateral flow is used to conduct visible moieties, especially labeled particles, e.g., dyed latex, red blood cells or liposomes capable of reacting with analyte or a competitor thereto into a capture zone for detection, using a bibulous support made nonbibulous by treatment with a blocking agent. The result is a one-step assay which can be conducted in a very short period of time (typically, within 60 seconds), and wherein the readout is usually available instantaneously upon the sample contacting a capture zone.
These one-step assays are complex devices containing a number of immunoassay reagents. Because the ability to manipulate the sample is restricted, it is desirable to develop other design variations that increase the range of sensitivity of the assay without increasing either the time necessary to perform the assay or the number of false positive results.
None of the references described herein is admitted to be prior art.